Programmable Computational RNA Droplets Assembled via Kissing-Loop Interaction.

DNA droplets, artificial liquid-like condensates of well-engineered DNA sequences, allow the critical aspects of phase-separated biological condensates to be harnessed programmably, such as molecular sensing and phase-state regulation. In contrast, their RNA-based counterparts remain less explored despite more diverse molecular structures and functions ranging from DNA-like to protein-like features. Here, we design and demonstrate computational RNA droplets capable of two-input AND logic operations. We use a multibranched RNA nanostructure as a building block comprising multiple single-stranded RNAs. Its branches engaged in RNA-specific kissing-loop (KL) interaction enables the self-assembly into a network-like microstructure. Upon two inputs of target miRNAs, the nanostructure is programmed to break up into lower-valency structures that are interconnected in a chain-like manner. We optimize KL sequences adapted from viral sequences by numerically and experimentally studying the base-wise adjustability of the interaction strength. Only upon receiving cognate microRNAs, RNA droplets selectively show a drastic phase-state change from liquid to dispersed states due to dismantling of the network-like microstructure. This demonstration strongly suggests that the multistranded motif design offers a flexible means to bottom-up programming of condensate phase behavior. Unlike submicroscopic RNA-based logic operators, the macroscopic phase change provides a naked-eye-distinguishable readout of molecular sensing. Our computational RNA droplets can be applied to in situ programmable assembly of computational biomolecular devices and artificial cells from transcriptionally derived RNA within biological/artificial cells.

Fibrinopeptide A-induced blood-feeding arrest in the yellow fever mosquito Aedes aegypti.

Female mosquitoes engage in blood feeding from their hosts to facilitate egg maturation but cease feeding once a sufficient blood meal has been acquired. Abdominal distention has been proposed as a contributing factor; however, it has also been suggested that there are chemical controls. In this study, we focus on negative chemical regulators of blood feeding, particularly those present in the host blood. Serum derived from animal blood inhibits the feeding of ATP, a phagostimulant of blood feeding in Aedes aegypti. Fibrinopeptide A (FPA), a 16-amino acid peptide cleaved from fibrinogen during blood coagulation, serves as an inhibitory factor in the serum. Our findings suggest that blood-feeding arrest in female mosquitoes is triggered by the detection of FPA in the host blood, which increases as blood coagulation proceeds in the mosquito's midgut, highlighting the role of host-derived substances as negative regulators of mosquito behavior.

Development of PCR primers enabling the design of flexible sticky ends for efficient concatenation of long DNA fragments.

We developed chemically modified PCR primers that allow the design of flexible sticky ends by introducing a photo-cleavable group at the phosphate moiety. Nucleic acid derivatives containing o-nitrobenzyl photo-cleavable groups with a tert-butyl group at the benzyl position were stable during strong base treatment for oligonucleotide synthesis and thermal cycling in PCR reactions. PCR using primers incorporating these nucleic acid derivatives confirmed that chain extension reactions completely stopped at position 1 before and after the site of the photo-cleavable group was introduced. DNA fragments of 2 and 3 kbp, with sticky ends of 50 bases, were successfully concatenated with a high yield of 77%. A plasmid was constructed using this method. Finally, we applied this approach to construct a 48.5 kbp lambda phage DNA, which is difficult to achieve using restriction enzyme-based methods. After 7 days, we were able to confirm the generation of DNA of the desired length. Although the efficiency is yet to be improved, the chemically modified PCR primer offers potential to complement enzymatic methods and serve as a DNA concatenation technique.

Ebola virus-like particles reprogram cellular metabolism.

Ebola virus can trigger a release of pro-inflammatory cytokines with subsequent vascular leakage and impairment of clotting finally leading to multiorgan failure and shock after entering and infecting patients. Ebola virus is known to directly target endothelial cells and macrophages, even without infecting them, through direct interactions with viral proteins. These interactions affect cellular mechanics and immune processes, which are tightly linked to other key cellular functions such as metabolism. However, research regarding metabolic activity of these cells upon viral exposure remains limited, hampering our understanding of its pathophysiology and progression. Therefore, in the present study, an untargeted cellular metabolomic approach was performed to investigate the metabolic alterations of primary human endothelial cells and M1 and M2 macrophages upon exposure to Ebola virus-like particles (VLP). The results show that Ebola VLP led to metabolic changes among endothelial, M1, and M2 cells. Differential metabolite abundance and perturbed signaling pathway analysis further identified specific metabolic features, mainly in fatty acid-, steroid-, and amino acid-related metabolism pathways for all the three cell types, in a host cell specific manner. Taken together, this work characterized for the first time the metabolic alternations of endothelial cells and two primary human macrophage subtypes after Ebola VLP exposure, and identified the potential metabolites and pathways differentially affected, highlighting the important role of those host cells in disease development and progression. KEY MESSAGES: • Ebola VLP can lead to metabolic alternations in endothelial cells and M1 and M2 macrophages. • Differential abundance of metabolites, mainly including fatty acids and sterol lipids, was observed after Ebola VLP exposure. • Multiple fatty acid-, steroid-, and amino acid-related metabolism pathways were observed perturbed.

Dried human-cultured epidermis accelerates wound healing in a porcine partial-thickness skin defect model.

Introduction: Autologous cultured epidermis (CE) is an effective approach for overcoming the deficiency of donor sites to treat extensive burns. However, the production of autologous CE takes 3-4 weeks, which prevents its use during the life-threatening period of severe burns. In contrast, allogeneic CE can be prepared in advance and used as a wound dressing, releasing several growth factors stimulating the activity of recipient cells at the application site. Dried CE is prepared by drying CEs under controlled temperature and humidity conditions until all the water is completely removed and no viable cells are present. Dried CE accelerates wound healing in a murine skin defect model and is potentially a new therapeutic strategy. However, the dried CE safety and efficacy have not yet been studied in large animal models. Therefore, we studied the safety and efficacy of human-dried CE in wound healing using a miniature swine model. Methods: Human CE was manufactured using Green's method from donor keratinocytes. Three types of CEs (Fresh, Cryopreserved, and Dried) were prepared, and the ability of each CE to promote keratinocyte proliferation was confirmed in vitro. Extracts of the three CEs were added to keratinocytes seeded in 12-well plates, and cell proliferation was evaluated using the WST-8 assay for 7 days. Next, we prepared a partial-thickness skin defect on the back of a miniature swine and applied three types of human CE to evaluate wound healing promotion. On days 4 and 7, the specimens were harvested for hematoxylin-eosin, AZAN, and anti-CD31 staining to assess epithelialization, granulation tissue, and capillary formation. Results: The conditioned medium containing dried CE extract significantly enhanced keratinocyte proliferation compared to the control group (P < 0.05). In vivo experiments revealed that human-dried CE significantly accelerated epithelialization at day 7 to the same extent as fresh CE, compared to the control group (P < 0.05). The three CE groups similarly affected granulation formation and neovascularization. Conclusions: Dried CE accelerated epithelialization in a porcine partial-thickness skin defect model, suggesting that it may be an effective burn treatment alternative. A clinical study with a long-term follow-up is needed to assess the applicability of CEs in clinics.

Establishment of a keratinocyte and fibroblast bank for clinical applications in Japan.

Regenerative medicine products using allogeneic cells, such as allogeneic cultured epidermis (allo-CE), have become a more critical therapeutic method for the treatment of burns. However, there are no clinically available allo-CE products in Japan. Therefore, establishing a quality-controlled cell bank is mandatory to create regenerative medical products using allogeneic cells. In this study, we selected ten patients from the Department of Plastic Surgery of Kyoto University Hospital to become cell donors. We performed medical interviews and blood sampling for the donor to ensure virus safety. We examined the tissues and isolated cells by performing a nucleic acid test (NAT). To establish a master cell bank, quality evaluation was performed according to the International Conference of Harmonization (ICH) Q5A. Serological tests of the blood samples from the ten donors showed that two of them were ineligible. The cells registered in the cell bank were found to be compatible after virus testing was performed, and a master cell bank was constructed. Hence, we established a keratinocyte and fibroblast bank of clinically usable human cultured cells in Japan for the first time.

ePURE_JSBML: A Tool for Constructing a Deterministic Model of a Reconstituted Escherichia coli Protein Translation System with a User-Specified Nucleic Acid Sequence.

A protein synthesis system is one of the most important and complex biological networks, which translates DNA-encoded information into specific functions. Here, ePURE_JSBML, a tool for constructing biologically relevant large-scale and detailed computational models based on a reconstituted cell-free protein synthesis system, is presented; the user can specify the mRNA sequence, initial component concentration, and decoding rule. Model construction is based on Systems Biology Markup Language (SBML) using JSBML, a pure Java programming library. The tool generates simulation files, executable with Matlab, that enable a variety of simulation experiments including the synthesis of proteins of a few hundred residues.

A large-scale targeted proteomics of plasma extracellular vesicles shows utility for prognosis prediction subtyping in colorectal cancer.

PURPOSE: The pathogenesis of cancers depends on the molecular background of each individual patient. Therefore, verifying as many biomarkers as possible and clarifying their relationships with each disease status would be very valuable. We performed a large-scale targeted proteomics analysis of plasma extracellular vesicles (EVs) that may affect tumor progression and/or therapeutic resistance. EXPERIMENTAL DESIGN: Plasma EVs from 59 were collected patients with colorectal cancer (CRC) and 59 healthy controls (HC) in cohort 1, and 150 patients with CRC in cohort 2 for the large-scale targeted proteomics analysis of 457 proteins as candidate CRC markers. The Mann-Whitney-Wilcoxon test and random forest model were applied in cohort 1 to select promising markers. Consensus clustering was applied to classify patients with CRC in cohort 2. The Kaplan-Meier method and Cox regression analysis were performed to identify potential molecular factors contributing to the overall survival (OS) of patients. RESULTS: In the analysis of cohort 1, 99 proteins were associated with CRC. The analysis of cohort 2 revealed two clusters showing significant differences in OS (p = 0.017). Twelve proteins, including alpha-1-acid glycoprotein 1 (ORM1), were suggested to be associated with the identified CRC subtypes, and ORM1 was shown to significantly contribute to OS, suggesting that ORM1 might be one of the factors closely related to the OS. CONCLUSIONS: The study identified two novel subtypes of CRC, which exhibit differences in OS, as well as important biomarker proteins that are closely related to the identified subtypes. Liquid biopsy assessment with targeted proteomics analysis was proposed to be crucial for predicting the CRC prognosis.

A novel approach for wound treatment using dried cultured epidermal allograft: A phase I/II, single-center, open-label clinical trial.

BACKGROUND: Autologous cultured epidermis (CE) is successfully used in burn care, but it requires a manufacturing time of three weeks and is very expensive owing to its custom-made nature of treatment. To compensate this disadvantage, dried allogeneic CE promises a novel therapeutic approach; and previous reports have demonstrated its efficacy in promoting wound healing using a murine skin defect model. Herein, a prospective clinical study was conducted to confirm the safety and efficacy of dried allogeneic CE for wound treatment. METHODS: Dried CE was manufactured using donor keratinocytes obtained from excess surgical skin and applied to skin defects that were at least 3 cm in length and less than 10 % of the body surface area of the patients. The patients were observed for 14 days after CE application. The primary endpoint was the incidence of adverse events and the secondary endpoint was the percentage of wound healed since baseline, on days 7 and 14. Furthermore, as a stratified analysis, the percentage of wound healed, specified as deep dermal burns, was calculated. RESULTS: Six patients (five burns and one skin ulcer after necrotizing fasciitis) enrolled in the study. As a serious adverse event, a local infection was observed in one patient, which resolved by debridement and conventional skin grafting. Other adverse events that were potentially related to this treatment included two cases of skin erosion, and one case of systemic fever. No unresolved adverse events remained at the end of the study period. The percentage of wound healed was 73.4 ± 19.2 % on Day 7, and 92.2 ± 11.8 % on Day 14. When the targeted disease was restricted to deep dermal burns, the percentage of wound healed was 69.9 ± 28.9 % on Day 7 and 90.5 ± 13.2 % on Day 14. CONCLUSION: Treatment with dried CE was safely performed without any unresolved severe adverse effects. Dried CE is a new and promising modality for skin defect treatment, such as burns and ulcers, and is expected to compensate for the disadvantages of autologous CE. However, large-scale clinical trials are required to confirm their efficacy.

Minimally Invasive Surgery for Colorectal Cancer During the COVID-19 Pandemic in a Tertiary Medical Facility in Japan.

BACKGROUND/AIM: The coronavirus disease 2019 (COVID-19) pandemic has reduced hospital visits due to concerns regarding infection and also resulted in cancer screening delays. These changes may have had an impact on the progression of colorectal cancer (CRC). Therefore, the present study investigated the effects of the COVID-19 pandemic on minimally invasive surgery (MIS) for CRC using a correlation analysis of clinical outcomes before and during the COVID-19 pandemic. PATIENTS AND METHODS: The present study targeted CRC patients who underwent MIS between January 2018 and December 2019 (pre-COVID-19) and between April 2020 and March 2021 (COVID-19). A comparison analysis of clinical, surgical, and pathological findings between the pre-COVID-19 and COVID-19 groups was performed. RESULTS: Ninety-one patients underwent MIS for CRC pre-COVID-19 and 67 during COVID-19. The number of CRC cases detected by fecal occult blood tests was slightly higher in the pre-COVID-19 group than that in the COVID-19 group. Re-evaluations of laparoscopic videos revealed that the number of cases of surgical T4 CRC resected with the combined resection of the adjacent organs was significantly higher in the COVID-19 group than that in the pre-COVID-19 group (16.4 vs. 4.4%, p=0.010). Furthermore, surgical times were significantly longer in the COVID-19 group than those in the pre-COVID-19 group (p<0.001). Pathological findings showed that the number of pT4 cases was significantly higher in the COVID-19 group than that in the pre-COVID-19 group (p=0.026). CONCLUSION: The number of T4 CRC cases was higher during than before the COVID-19 pandemic, with increases in the surgical difficulty of MIS.

Transfer RNA Synthesis-Coupled Translation and DNA Replication in a Reconstituted Transcription/Translation System.

Transfer RNAs (tRNAs) are key molecules involved in translation. In vitro synthesis of tRNAs and their coupled translation are important challenges in the construction of a self-regenerative molecular system. Here, we first purified EF-Tu and ribosome components in a reconstituted translation system of Escherichia coli to remove residual tRNAs. Next, we expressed 15 types of tRNAs in the repurified translation system and performed translation of the reporter luciferase gene depending on the expression. Furthermore, we demonstrated DNA replication through expression of a tRNA encoded by DNA, mimicking information processing within the cell. Our findings highlight the feasibility of an in vitro self-reproductive system, in which tRNAs can be synthesized from replicating DNA.

A red light-responsive photoswitch for deep tissue optogenetics.

Red light penetrates deep into mammalian tissues and has low phototoxicity, but few optogenetic tools that use red light have been developed. Here we present MagRed, a red light-activatable photoswitch that consists of a red light-absorbing bacterial phytochrome incorporating a mammalian endogenous chromophore, biliverdin and a photo-state-specific binder that we developed using Affibody library selection. Red light illumination triggers the binding of the two components of MagRed and the assembly of split-proteins fused to them. Using MagRed, we developed a red light-activatable Cre recombinase, which enables light-activatable DNA recombination deep in mammalian tissues. We also created red light-inducible transcriptional regulators based on CRISPR-Cas9 that enable an up to 378-fold activation (average, 135-fold induction) of multiple endogenous target genes. MagRed will facilitate optogenetic applications deep in mammalian organisms in a variety of biological research areas.

Capillary microsampling-based single-cell metabolomics by mass spectrometry and its applications in medicine and drug discovery.

Characterization of cellular metabolic states is a technical challenge in biomedicine. Cellular heterogeneity caused by inherent diversity in expression of metabolic enzymes or due to sensitivity of metabolic reactions to perturbations, necessitates single cell analysis of metabolism. Heterogeneity is typically seen in cancer and thus, single-cell metabolomics is expectedly useful in studying cancer progression, metastasis, and variations in cancer drug response. However, low sample volumes and analyte concentrations limit detection of critically important metabolites. Capillary microsampling-based mass spectrometry approaches are emerging as a promising solution for achieving single-cell omics. Herein, we focus on the recent advances in capillary microsampling-based mass spectrometry techniques for single-cell metabolomics. We discuss recent technical developments and applications to cancer medicine and drug discovery.

Tracking metabolites at single-cell resolution reveals metabolic dynamics during plant mitosis.

Analyzing only one cell allows the changes and characteristics of intracellular metabolites during the chromosome segregation process to be precisely captured and mitotic sub-phases to be dissected at the metabolite level.

Dried human cultured epidermis accelerates wound healing in diabetic mouse skin defect wounds.

Cryopreserved allogeneic cultured epidermis (CE) is used for treating second-degree burn wounds and diabetic foot ulcers; however, the need for cryopreservation limits its use. We have previously reported that CE accelerates wound healing irrespective of its viability and hypothesized that dehydrated CEs lacking living cells may act as an effective wound dressing. We prepared dried CE and investigated its morphological and physical properties and wound-healing effects and compared them with those of cryopreserved CE. Hematoxylin-eosin staining, immunostaining for basement membrane, and electron microscopy revealed that the morphologies of dried CE and cryopreserved CE were comparable and that the membrane structure was not damaged. The breaking strength, modulus of elasticity, and water permeability of dried CE were comparable with those of the cryopreserved CE. Furthermore, the levels of various active cytokines and chemokines in dried CE were comparable with those in cryopreserved CE. Dried CE applied to skin defect in diabetic mice significantly reduced the wound area and increased the new epithelium length 4 and 7 days after implantation, similar to that observed for cryopreserved CE. Consequently, dried CE had similar morphological and physical properties and wound-healing effects compared with those of cryopreserved CE and can be a physiological and versatile wound-dressing.

Cell-Free Synthesis of Human Endothelin Receptors and Its Application to Ribosome Display.

Engineering G-protein-coupled receptors (GPCRs) for improved stability or altered function is of great interest, as GPCRs consist of the largest protein family, are involved in many important signaling pathways, and thus, are one of the major drug targets. Here, we report the development of a high-throughput screening method for GPCRs using a reconstituted in vitro transcription-translation (IVTT) system. Human endothelin receptor type-B (ETBR), a class A GPCR that binds endothelin-1 (ET-1), a 21-residue peptide hormone, was synthesized in the presence of nanodisc (ND) composed of a phospholipid, 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG). The ET-1 binding of ETBR was significantly reduced or was undetectable when other phospholipids were used for ND preparation. However, when functional ETBR purified from Sf9 cells was reconstituted into NDs, ET-1 binding was observed with two different phospholipids tested, including POPG. These results suggest that POPG likely supports the folding of ETBR into its functional form in the IVTT system. Using the same conditions as ETBR, whose three-dimensional structure has been solved, human endothelin receptor type-A (ETAR), whose three-dimensional structure remains unsolved, was also synthesized in its functional form. By adding POPG-ND to the IVTT system, both ETAR and ETBR were successfully subjected to ribosome display, a method of in vitro directed evolution that facilitates the screening of up to 1012 mutants. Finally, using a mock library, we showed that ribosome display can be applied for gene screening of ETBR, suggesting that high-throughput screening and directed evolution of GPCRs is possible in vitro.

Efficient and Precise Protein Synthesis in a Cell-Free System Using a Set of In Vitro Transcribed tRNAs with Nucleotide Modifications.

Reconstitution of a complicated system with a minimal set of components is essential for understanding the mechanisms of how the input is reflected in the output, which is fundamental for further engineering of the corresponding system. We have recently developed a reconstituted cell-free protein synthesis system equipped only with 21 in vitro transcribed tRNAs, one of the minimal systems for understanding the genetic code decoding mechanisms. Introduction of several nucleotide modifications to the transcribed tRNAs showed improvement of both protein synthesis efficiency and its fidelity, suggesting various combinations of tRNAs and their modifications can be evaluated in the developed system. In this chapter, we describe how to prepare this minimal system. Methods for preparing the transcribed tRNAs, their modifications, and the protein production using the set of prepared tRNAs are shown.

Versatile and multiplexed mass spectrometry-based absolute quantification with cell-free-synthesized internal standard peptides.

Preparation of stable isotope-labeled internal standard peptides is crucial for mass spectrometry (MS)-based targeted proteomics. Herein, we developed versatile and multiplexed absolute protein quantification method using MS. A previously developed method based on the cell-free peptide synthesis system, termed MS-based quantification by isotope-labeled cell-free products (MS-QBiC), was improved for multiple peptide synthesis in one-pot reaction. We pluralized the quantification tags used for the quantification of synthesized peptides and thus, made it possible to use cell-free synthesized isotope-labeled peptides as mixtures for the absolute quantification. The improved multiplexed MS-QBiC method was proved to be applied to clarify ribosomal proteins stoichiometry in the ribosomal subunit, one of the largest cellular complexes. The study demonstrates that the developed method enables the preparation of several dozens and even several hundreds of internal standard peptides within a few days for quantification of multiple proteins with only a single-run of MS analysis. SIGNIFICANCE: The developed method can be applied for the preparation of internal standard peptides without limiting the number of peptides to be synthesized, which may result in more practical screening of quantitatively reliable peptides, one of the fundamental steps in the reliable absolute quantification using MS. Furthermore, the method is highly versatile for proteome analysis of any organisms or species without any cDNA or SIL peptide libraries. The quantification can be finished in a few days including design and preparation of appropriate SIL peptides using small-scale batch cell-free reactions, which has a potential to be a part of the standard methodology in a field of quantitative proteomics.

In vitro reconstitution of the Escherichia coli 70S ribosome with a full set of recombinant ribosomal proteins.

Many studies of the reconstitution of the Escherichia coli small ribosomal subunit from its individual molecular parts have been reported, but contrastingly, similar studies of the large ribosomal subunit have not been well performed to date. Here, we describe protocols for preparing the 33 ribosomal proteins of the E. coli 50S subunit and demonstrate successful reconstitution of a functionally active 50S particle that can perform protein synthesis in vitro. We also successfully reconstituted both ribosomal subunits (30S and 50S) and 70S ribosomes using a full set of recombinant ribosomal proteins by integrating our developed method with the previously developed fully recombinant-based integrated synthesis, assembly and translation. The approach described here makes a major contribution to the field of ribosome engineering and could be fundamental to the future studies of ribosome assembly processes.

Antisense Oligonucleotide Modified with Disulfide Units Induces Efficient Exon Skipping in mdx Myotubes through Enhanced Membrane Permeability and Nucleus Internalization.

We have found that antisense oligonucleotides and siRNA molecules modified with repeat structures of disulfide units can be directly introduced into the cytoplasm and exhibit a suppressive effect on gene expression. In this study, we analyzed the mechanism of cellular uptake of these membrane-permeable oligonucleotides (MPONs). Time-course analysis by confocal microscopy showed that the uptake of MPONs from the plasma membrane to the cytoplasm reached 50 % of the total uptake in about 5 min. In addition, analysis of the plasma membrane proteins to which MPONs bind, identified several proteins, including voltage-dependent anion channel. Next, we analyzed the behavior of MPONs in the cell and found them to be abundant in the nucleus as early as 24 h after addition with the amount increasing further after 48 and 72 h. The amount of MPONs was 2.5-fold higher than that of unmodified oligonucleotides in the nucleus after 72 h. We also designed antisense oligonucleotides and evaluated the effect of MPONs on mRNA exon skipping using DMD model cells; MPONs caused exon skipping with 69 % efficiency after 72 h, which was three times higher than the rate of the control. In summary, the high capacity for intracytoplasmic and nuclear translocation of MPONs is expected to be useful for therapeutic strategies targeting exon skipping.